Chemical and biochemical analyses are performed in large numbers in chemical, pharmaceutical and clinical laboratories, and technicians in these laboratories see an ever increasing need to shorten the time required for a given analysis. Some of the reasons relate to the cost of labor and the need for the availability of valuable laboratory space and equipment. Other reasons arise from the fact that many substances studied in these analyses are labile, such as substances taking part in fast chemical reactions, including biochemical coupling reactions, and others such as radiolabeled compounds have a short lifetime.
One of the most widely used analytical techniques is electrophoresis, owing to its versatility and its ability to separate and identify the components of complex chemical and biochemical mixtures. The variety of different forms of electrophoresis which are now in use has extended the applicability of the method to analyses of many different types of molecules, ranging from simple organic molecules to macromolecules such as polypeptides, enzymes, blood factors and other biological compounds, and nucleic acids. The development of capillary electrophoresis has further extended the usefulness of the method by permitting analyses to be performed on extremely small samples, and by permitting the electrophoretic media to tolerate high voltages and thereby achieve even shorter separation times and more complex separations. Electrophoresis is commonly performed in capillaries at voltages of 100-300 V/cm without thermally induced zone deformation which can cause a significant loss of resolution. In electrophoresis cells other than capillaries, typical voltages are 10-100 V/cm.
In capillaries as well as in tube gels, slab gels and other configurations of the electrophoresis media, laboratories would benefit from still further decreases in the time required to perform electrophoresis. One logical way to do this would be to increase the rate of migration for individual solutes. The time required for the migration of a solute to a detector is inversely proportional to the field strength, as shown by the following equation: ##EQU1## where: t is the migration time,
L is the migration distance to the detector, PA1 u is the mobility of the solute (i.e., the velocity of the solute divided by the field strength, and PA1 F is the electrical field strength. PA1 L' is the length of the capillary tube, PA1 I is the current in amperes, and PA1 R is the inner radius of the capillary in centimeters. PA1 (1) buffering agents with a small number of charged groups per molecule, and preferably of a relatively high molecular weight; PA1 (2) carrier ampholytes fractionated to a narrow pH range by isoelectric focusing; PA1 (3) low molecular weight buffering ampholytes at their isoelectric points, the isoelectric point being one which is close in value to one of the pK values of the ampholyte; and PA1 (4) high molecular weight buffering ampholytes in which the acidic and basic groups have the same or very close pK values.
Increasing the field strength would clearly decrease the migration times, but high field strengths are usually accompanied by high Joule heating, which leads to zone deformation and consequently a decrease in solute resolution. High field strengths also cause bubbles of water vapor to form. These bubbles interfere with electrophoretic migration and obscure the resolution of the zones.